ß-Aminopropionitrile Induces Distinct Pathologies in the Ascending and Descending Thoracic Aortic Regions of Mice.

BACKGROUND: ß-aminopropionitrile (BAPN) is a pharmacological inhibitor of LOX (lysyl oxidase) and LOXLs (LOX-like proteins). Administration of BAPN promotes aortopathies, although there is a paucity of data on experimental conditions to generate pathology. The objective of this study was to define experimental parameters and determine whether equivalent or variable aortopathies were generated throughout the aortic tree during BAPN administration in mice. METHODS: BAPN was administered in drinking water for a period ranging from 1 to 12 weeks. The impacts of BAPN were first assessed with regard to BAPN dose, and mouse strain, age, and sex. BAPN-induced aortic pathological characterization was conducted using histology and immunostaining. To investigate the mechanistic basis of regional heterogeneity, the ascending and descending thoracic aortas were harvested after 1 week of BAPN administration before the appearance of overt pathology. RESULTS: BAPN-induced aortic rupture predominantly occurred or originated in the descending thoracic aorta in young C57BL/6J or N mice. No apparent differences were found between male and female mice. For mice surviving 12 weeks of BAPN administration, profound dilatation was consistently observed in the ascending region, while there were more heterogeneous changes in the descending thoracic region. Pathological features were distinct between the ascending and descending thoracic regions. Aortic pathology in the ascending region was characterized by luminal dilatation and elastic fiber disruption throughout the media. The descending thoracic region frequently had dissections with false lumen formation, collagen deposition, and remodeling of the wall surrounding the false lumen. Cells surrounding the false lumen were predominantly positive for α-SMA (α-smooth muscle actin). One week of BAPN administration compromised contractile properties in both regions equivalently, and RNA sequencing did not show obvious differences between the 2 aortic regions in smooth muscle cell markers, cell proliferation markers, and extracellular components. CONCLUSIONS: BAPN-induced pathologies show distinct, heterogeneous features within and between ascending and descending aortic regions in mice.

Functional Exploration of Conserved Sequences in the Distal Face of Angiotensinogen-Brief Report.

BACKGROUND: Angiotensinogen (AGT) is an essential component in the renin-angiotensin system. AGT has highly conserved sequences in the loop and ß-sheet regions among species; however, their functions have not been studied. METHODS: Adeno-associated viral vector (AAV) serotype 2/8 encoding mouse AGT with mutations of conserved sequences in the loop (AAV.loop-Mut), ß-sheet (AAV.ßsheet-Mut), or both regions (AAV.loop/ßsheet-Mut) was injected into male hepatocyte-specific AGT-deficient (hepAGT-/-) mice in an LDL (low-density lipoprotein) receptor-deficient background. AAV containing mouse wild-type AGT (AAV.mAGT) or a null vector (AAV.null) were used as controls. Two weeks after AAV administration, all mice were fed a western diet for 12 weeks. To determine how AGT secretion is regulated in hepatocytes, AAVs containing the above mutations were transducted into HepG2 cells. RESULTS: In hepAGT-/- mice infected with AAV.loop-Mut or ßsheet-Mut, plasma AGT concentrations, systolic blood pressure, and atherosclerosis were comparable to those in AAV.mAGT-infected mice. Interestingly, plasma AGT concentrations, systolic blood pressure, and atherosclerotic lesion size in hepAGT-/- mice infected with AAV.loop/ßsheet-Mut were not different from mice infected with AAV.null. In contrast, hepatic Agt mRNA abundance was elevated to a comparable magnitude as AAV.mAGT-infected mice. Immunostaining showed that AGT protein was accumulated in hepatocytes of mice infected with AAV.loop/ßsheet-Mut or HepG2 cells transducted with AAV.loop/ßsheet-Mut. Accumulated AGT was not located in the endoplasmic reticulum. CONCLUSIONS: The conserved sequences in either the loop or ß-sheet region individually have no effect on AGT regulation, but the conserved sequences in both regions synergistically contribute to the secretion of AGT from hepatocytes.

Second Heart Field-Derived Cells Contribute to Angiotensin II-Mediated Ascending Aortopathies.

BACKGROUND: The ascending aorta is a common location for aneurysm and dissection. This aortic region is populated by a mosaic of medial and adventitial cells that are embryonically derived from either the second heart field (SHF) or the cardiac neural crest. SHF-derived cells populate areas that coincide with the spatial specificity of thoracic aortopathies. The purpose of this study was to determine whether and how SHF-derived cells contribute to ascending aortopathies. METHODS: Ascending aortic pathologies were examined in patients with sporadic thoracic aortopathies and angiotensin II (AngII)-infused mice. Ascending aortas without overt pathology from AngII-infused mice were subjected to mass spectrometry-assisted proteomics and molecular features of SHF-derived cells were determined by single-cell transcriptomic analyses. Genetic deletion of either Lrp1 (low-density lipoprotein receptor-related protein 1) or Tgfbr2 (transforming growth factor-ß receptor type 2) in SHF-derived cells was conducted to examine the effect of SHF-derived cells on vascular integrity. RESULTS: Pathologies in human ascending aortic aneurysmal tissues were predominant in outer medial layers and adventitia. This gradient was mimicked in mouse aortas after AngII infusion that was coincident with the distribution of SHF-derived cells. Proteomics indicated that brief AngII infusion before overt pathology occurred evoked downregulation of smooth muscle cell proteins and differential expression of extracellular matrix proteins, including several LRP1 ligands. LRP1 deletion in SHF-derived cells augmented AngII-induced ascending aortic aneurysm and rupture. Single-cell transcriptomic analysis revealed that brief AngII infusion decreased Lrp1 and Tgfbr2 mRNA abundance in SHF-derived cells and induced a unique fibroblast population with low abundance of Tgfbr2 mRNA. SHF-specific Tgfbr2 deletion led to embryonic lethality at E12.5 with dilatation of the outflow tract and retroperitoneal hemorrhage. Integration of proteomic and single-cell transcriptomics results identified PAI1 (plasminogen activator inhibitor 1) as the most increased protein in SHF-derived smooth muscle cells and fibroblasts during AngII infusion. Immunostaining revealed a transmural gradient of PAI1 in both ascending aortas of AngII-infused mice and human ascending aneurysmal aortas that mimicked the gradient of medial and adventitial pathologies. CONCLUSIONS: SHF-derived cells exert a critical role in maintaining vascular integrity through LRP1 and transforming growth factor-ß signaling associated with increases of aortic PAI1.

Inhibition of the Renin-Angiotensin System Fails to Suppress ß-Aminopropionitrile-Induced Thoracic Aortopathy in Mice-Brief Report.

BACKGROUND: Cross-linking of lysine residues in elastic and collagen fibers is a vital process in aortic development. Inhibition of lysyl oxidase by BAPN (ß-aminopropionitrile) leads to thoracic aortopathies in mice. Although the renin-angiotensin system contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the renin-angiotensin system protects against aortopathy caused by the impairment of elastic fiber/collagen crosslinking. METHODS: BAPN (0.5% wt/vol) was started in drinking water to induce aortopathies in male C57BL/6J mice at 4 weeks of age for 4 weeks. Five approaches were used to investigate the impact of the renin-angiotensin system. Bulk RNA sequencing was performed to explore potential molecular mechanisms of BAPN-induced thoracic aortopathies. RESULTS: Losartan increased plasma renin concentrations significantly, compared with vehicle-infused mice, indicating effective angiotensin II type 1 receptor inhibition. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor of the renin-angiotensin system, irbesartan, an insurmountable inhibitor, was also tested. Although increased plasma renin concentrations indicated effective inhibition, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin II type 1 receptor blockade. Next, we inhibited angiotensin II production by pharmacological or genetic depletion of AGT (angiotensinogen), the unique precursor of angiotensin II. However, neither suppressed BAPN-induced thoracic aortic rupture and dilatation. Aortic RNA sequencing revealed molecular changes during BAPN administration that were distinct from other types of aortopathies in which angiotensin II type 1 receptor inhibition protects against aneurysm formation. CONCLUSIONS: Inhibition of either angiotensin II action or production of the renin-angiotensin system does not attenuate BAPN-induced thoracic aortopathies in mice.

Inducible Depletion of Calpain-2 Mitigates Abdominal Aortic Aneurysm in Mice.

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Deletion of AT1a (Angiotensin II Type 1a) Receptor or Inhibition of Angiotensinogen Synthesis Attenuates Thoracic Aortopathies in Fibrillin1C1041G/+ Mice.

Objective: A cardinal feature of Marfan syndrome is thoracic aortic aneurysm. The contribution of the renin-angiotensin system via AT1aR (Ang II [angiotensin II] receptor type 1a) to thoracic aortic aneurysm progression remains controversial because the beneficial effects of angiotensin receptor blockers have been ascribed to off-target effects. This study used genetic and pharmacological modes of attenuating angiotensin receptor and ligand, respectively, to determine their roles on thoracic aortic aneurysm in mice with fibrillin-1 haploinsufficiency (Fbn1C1041G/+). Approach and Results: Thoracic aortic aneurysm in Fbn1C1041G/+ mice was found to be strikingly sexual dimorphic. Males displayed aortic dilation over 12 months while aortic dilation in Fbn1C1041G/+ females did not differ significantly from wild-type mice. To determine the role of AT1aR, Fbn1C1041G/+ mice that were either +/+ or -/- for AT1aR were generated. AT1aR deletion reduced expansion of ascending aorta and aortic root diameter from 1 to 12 months of age in males. Medial thickening and elastin fragmentation were attenuated. An antisense oligonucleotide against angiotensinogen was administered to male Fbn1C1041G/+ mice to determine the effects of Ang II depletion. Antisense oligonucleotide against angiotensinogen administration attenuated dilation of the ascending aorta and aortic root and reduced extracellular remodeling. Aortic transcriptome analyses identified potential targets by which inhibition of the renin-angiotensin system reduced aortic dilation in Fbn1C1041G/+ mice. Conclusions: Deletion of AT1aR or inhibition of Ang II production exerted similar effects in attenuating pathologies in the proximal thoracic aorta of male Fbn1C1041G/+ mice. Inhibition of the renin-angiotensin system attenuated dysregulation of genes within the aorta related to pathology of Fbn1C1041G/+ mice.

Two Amino Acids Proximate to the Renin Cleavage Site of Human Angiotensinogen Do Not Affect Blood Pressure and Atherosclerosis in Mice-Brief Report.

OBJECTIVE: Renin cleavage of angiotensinogen has species specificity. As the residues at positions 11 and 12 are different between human angiotensinogen and mouse angiotensinogen, we determined whether these 2 residues in angiotensinogen affect renin cleavage and angiotensin II-mediated blood pressure regulation and atherosclerosis using an adenoassociated viral approach for manipulating angiotensinogen in vivo. Approach and Results: Hepatocyte-specific angiotensinogen deficient (hepAGT-/-) mice in an LDL receptor-deficient background were infected with adenoassociated virals containing a null insert, human angiotensinogen, or mouse angiotensinogen expressing the same residues of the human protein at positions 11 and 12 (mouse angiotensinogen [L11V;Y12I]). Expression of human angiotensinogen in hepAGT-/- mice led to high plasma human angiotensinogen concentrations without changes in plasma endogenous mouse angiotensinogen, plasma renin concentrations, blood pressure, or atherosclerosis. This is consistent with human angiotensinogen not being cleaved by mouse renin. To determine whether the residues at positions 11 and 12 in human angiotensinogen lead to the inability of mouse renin to cleave human angiotensinogen, hepAGT-/- mice were injected with adenoassociated viral vector encoding mouse angiotensinogen (L11V;Y12I). Expression of mouse angiotensinogen (L11V;Y12I) in hepAGT-/- mice resulted in increased plasma mouse angiotensinogen concentrations, reduced renin concentrations, and increased renal AngII concentrations that were comparable to their concentrations in hepAGT+/+ mice. This mouse angiotensinogen variant increased blood pressure and atherosclerosis in hepAGT-/- mice to the magnitude of hepAGT+/+ mice. CONCLUSIONS: Replacement of L11 and Y12 to V11 and I12, respectively, in mouse angiotensinogen does not affect renin cleavage, blood pressure, and atherosclerosis in LDL receptor-deficient mice.

SR-BI (Scavenger Receptor BI), Not LDL (Low-Density Lipoprotein) Receptor, Mediates Adrenal Stress Response-Brief Report.

OBJECTIVE: Adrenal gland secretes stress-induced glucocorticoids (iGCs) to coping with stress. Previous study showed that SR-BI (scavenger receptor BI) null (SR-BI-/-) mice failed to generate iGC in stress conditions, suggesting that SR-BI-mediated cholesterol uptake from HDL (high-density lipoprotein) is a key regulator for iGC production. However, the LDL (low-density lipoprotein)/LDLr (LDL receptor) pathway can also provide cholesterol for iGC synthesis, but rodents have limited LDL levels in circulation. Here, we generated SR-BI-/-ApoBtg (apolipoprotein B transgenic) mice with normal LDL levels in circulation to determine the relative contribution of the HDL/SR-BI and LDL/LDLr pathways to iGC production in stress conditions. Approach and Results: To obtain mouse models with normal LDL levels, SR-BI-/- mice were bred to ApoBtg mice. Then, the F1 SR-BI±ApoBtg mice were backcrossed to SR-BI-/- to obtain SR-BI-/-ApoBtg, SR-BI-/-ApoBwt (apolipoprotein B wild type), and SR-BI+/+ApoBtg mice. We first examined the lipoprotein profile, which shows a 6.5-fold increase in LDL levels in SR-BI-/-ApoBtg mice compared with SR-BI-/-ApoBwt mice. Then, we induced stress with adrenocorticotropic hormone and cecal ligation and puncture. One hour after adrenocorticotropic hormone stimulation, SR-BI+/+ApoBtg control mice produced iGC (14.9-fold), but both SR-BI-/-ApoBwt and SR-BI-/-ApoBtg showed no iGC production (P<0.001). Three hours after cecal ligation and puncture treatment, SR-BI+/+ApoBtg control mice showed iGC production (6.4-fold), but both SR-BI-/-ApoBwt and SR-BI-/-ApoBtg mice showed no iGC production (P<0.001). CONCLUSIONS: SR-BI-/-ApoBtg mice fail to produce iGC in stress conditions even though with restored LDL levels in circulation. These findings clarify that the HDL/SR-BI, not LDL/LDLr, pathway is responsible for iGC production in stress conditions.

Angiotensinogen and Megalin Interactions Contribute to Atherosclerosis-Brief Report.

Objective- AGT (Angiotensinogen) is the unique precursor of the renin-angiotensin system that is sequentially cleaved by renin and ACE (angiotensin-converting enzyme) to produce Ang II (angiotensin II). In this study, we determined how these renin-angiotensin components interact with megalin in kidney to promote atherosclerosis. Approach and Results- AGT, renin, ACE, and megalin were present in the renal proximal convoluted tubules of wild-type mice. Hepatocyte-specific AGT deficiency abolished AGT protein accumulation in proximal tubules and diminished Ang II concentrations in kidney, while renin was increased. Megalin was most abundant in kidney and exclusively present on the apical side of proximal tubules. Inhibition of megalin by antisense oligonucleotides (ASOs) led to ablation of AGT and renin proteins in proximal tubules, while leading to striking increases of urine AGT and renin concentrations, and 70% reduction of renal Ang II concentrations. However, plasma Ang II concentrations were unaffected. To determine whether AGT and megalin interaction contributes to atherosclerosis, we used both male and female low-density lipoprotein receptor-/- mice fed a saturated fat-enriched diet and administered vehicles (PBS or control ASO) or megalin ASO. Inhibition of megalin did not affect plasma cholesterol concentrations, but profoundly reduced atherosclerotic lesion size in both male and female mice. Conclusions- These results reveal a regulatory role of megalin in the intrarenal renin-angiotensin homeostasis and atherogenesis, positing renal Ang II to be an important contributor to atherosclerosis that is mediated through AGT and megalin interactions.

Angiotensin I Infusion Reveals Differential Effects of Angiotensin-Converting Enzyme in Aortic Resident Cells on Aneurysm Formation.

BACKGROUND: Angiotensin (Ang)I is cleaved by angiotensin-converting enzyme (ACE) to generate AngII. The purpose of this study was to determine the roles of ACE in endothelial and smooth muscle cells in aortic aneurysms.Methods and Results:AngI infusion led to thoracic and abdominal aortic aneurysms in low-density lipoprotein receptor-deficient mice, which were ablated by ACE inhibition. Endothelial or smooth muscle cell-specific ACE deletion resulted in reduction of AngI-induced thoracic, but not abdominal, aortic dilatation. CONCLUSIONS: AngI infusion causes thoracic and abdominal aortic aneurysms in mice. ACE in aortic resident cells has differential effects on AngI-induced thoracic and abdominal aortic aneurysms.